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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Only bacteria-specific effects were noted in the bacteria reverse mutation assay, whereas the mutagenicity study in mammalian cells with the structural analogue was negative.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

The test substance did not induce DNA repair (as measured by unscheduled DNA synthesis) in rat liver nor did it induce micronuclei in the polychromatic erythrocytes of treated rats as seen in studies with close structural analogues.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Mode of Action Analysis / Human Relevance Framework

The test item Disperse Blue 366 was tested positivein the Bacteria Reverse Mutation Assay (Ames test) in Salmonella strains, but negative in an in-vivo micronucleus test in mice. A close structural analogue (Disperse Blue 291) was tested positive in an Ames test with nitroreductase and O-acetyltransferase positive Salmonella typhimurium strains, but negative with nitroreductase andO-acetyltransferase negative strains andin a test for unscheduled DNA synthesis. Another close structural analogue (Disperse Blue 165) was tested negative in a mutation assay in mammalian cells.

This positive effect in the bacterial mutation assay is a bacteria-specific effect due to bacterial nitro-reductases, which are highly effective in these bacterial strains, but not in mammalian cells.

The nitroreductase family comprises a group of flavin mononucleotide (FMN)- or flavin adenine dinucleotide (FAD) -dependent enzymes that are able to metabolize nitroaromatic and nitroheterocyclic derivatives (nitrosubstituted compounds) using the reducing power of nicotinamide adenine dinucleotide (NAD(P)H). These enzymes can be found in bacterial species and, to a lesser extent, in eukaryotes. The nitroreductase proteins play a central role in the activation of nitro-compounds. Type I nitroreductases can transfer two electrons from NAD(P)H to form the nitroso and hydroxylamino intermediates and finally the amino group. Type II nitroreductases transfer a single electron to the nitro group, forming a nitro anion radical, which in the presence of oxygen generates the superoxide anion in a futile redox cycle, regenerating the nitro group [de Oliveira et al. 2010].

The positive effect in the bacterial reverse mutation test (Ames) was clearly related to a bacteria-specific metabolism of the test substance, as it is well-known for aromatic nitro compounds to be positive in the Ames assay resulting from metabolism by the bacteria-specific enzyme nitro-reductase [Tweats et al. 2012]. This could be also be proved to be true in studies with Disperse Blue 291, which was tested for mutagenic activity in the Salmonella assay with strains with different levels of nitroreductase and O-acetyltransferase[Umbuzeiro et al. 2005]. In this study,Disperse Blue 291 showed mutagenic activity with all standard strains of Salmonella typhimurium tested (TA1537, TA1538, TA98 and TA100), except for TA1535.In nitroreductase and O-acetyltransferasenegative strains (TA98NR, TA98DNP6) not mutagenic activity was observed in the absence of S9, whereas themutagenic activity was increased with the nitroreductaseand/or O‑acetyltransferaseoverproducing strains, (YG1021, YG1024 and YG1041) This shows the importance of the bacterial acetyltransferase enzyme in the activation of Disperse Blue 291. Because of the remarkable increase in the response with the nitroreductase and O‑acetyltransferase overproducing strain (YG1041), it is assumed that the product of the nitroreductaseis a substrate for the O-acetyltransferase. As there was a very slight increase in mutagenicity with TA98NR, TA98, YG1021, TA98DNP6, and YG1024 in the presence of S9, it was assumed that P450 enzymes have also a role in the activation ofDisperse Blue 291, besides the bacterial enzymes. This could however not proven true in studies with Disperse Blue 165 in mammalian cells (HPRT assay) or in in-vivo studies with Disperse Blue 291 (UDS) or Disperse Blue 366 (MNT).

It has also been demonstrated in various other publications that this mutagenic activity is a bacteria-specific effect and that these Ames positive nitro-substances are not mutagenic in mammalian assays.

That the reduction of these nitro-compounds to mutagenic metabolites is a bacteria-specific effect is demonstrated in the following by means of the two compounds AMP397 and fexinidazole.

  • AMP397 is a drug candidate developed for the oral treatment of epilepsy. The molecule contains an aromatic nitro group, which obviously is a structural alert for mutagenicity. The chemical was mutagenic in Salmonellastrains TA97a, TA98 and TA100, all without S9, but negative in the nitroreductase-deficient strains TA98NR and TA100NR. Accordingly, the ICH standard battery mouse lymphomatkand mouse bone marrow micronucleus tests were negative, although a weak high toxicity-associated genotoxic activity was seen in a micronucleus test inV79 cells [Suter et al. 2002].The amino derivative of AMP397 was not mutagenic in wild type TA98 and TA100. To exclude that a potentially mutagenic metaboliteis released by intestinal bacteria, a MutaTMMouse study was done in colon and liver with five daily treatments at the MTD, and sampling of 3, 7 and 21 days post-treatment. No evidence of a mutagenic potential was found in colon and liver. Likewise, a comet assay did not detect any genotoxic activity in jejunum and liver of rats, after single treatment with a roughly six times higher dose than the transgenic study, which reflects the higher exposure observed in mice. In addition, a radioactive DNA binding assay in the liver of mice and rats did not find any evidence for DNA binding. Based on these results, it was concluded that AMP397 has no genotoxic potential in vivo. It was hypothesized that the positive Ames test was due to activation by bacterial nitro-reductase, as practically all mammalian assays including fourin vivoassays were negative, and no evidence for activation by mammalian nitro-reductase or other enzymes were seen. Furthermore, no evidence for excretion of metabolites mutagenic for intestinal cells by intestinal bacteria was found.
  • Fexinidazolewas in pre-clinical development as a broad-spectrum antiprotozoal drug by the Hoechst AG in the 1970s-1980s, but its clinical development was not pursued. Fexinidazole was rediscovered by the Drugs for Neglected Diseases initiative (DNDi) as drug candidate to cure the parasitic disease human African trypanomiasis (HAT), also known as sleeping sickness. The genotoxicity profile of fexinidazole, a 2-substituted 5-nitroimidazole, and its two active metabolites, the sulfoxide and sulfone derivatives were investigated [Tweats et al. 2012]. All the three compounds are mutagenic in the Salmonella/Ames test; however, mutagenicity is either attenuated or lost in Ames Salmonella strains that lack one or more nitroreductase(s). It is known that these enzymes can nitroreduce compounds with low redox potentials, whereas their mammalian cell counterparts cannot, under normal conditions. Fexinidazole and its metabolites have low redox potentials and all mammalian cell assays to detect genetic toxicity, conducted for this study either in vitro (micronucleus test in human lymphocytes) or in vivo (ex vivo unscheduled DNA synthesis in rats; bone marrow micronucleus test in mice), were negative.

Based on these data and the common mechanism between the reduction of these nitro-compounds, which is widely explored in literature [de Oliveira et al. 2010], it is concluded, that the mutagenic effects observed in the Ames test with Disperse Blue 366 is a bacteria specific effect and not relevant to mammalians.

Disperse Blue 366 and its structural analogues were not genotoxic in the mammalian in-vitro cell mutagenicity test (HPRT assay) and the in-vivo UDS and MNT test. Therefore, a direct genotoxic effect as well as a metabolisation towards genotoxic structures by mammalian species can be excluded.


De Oliveira IM, Bonatto D, Pega Henriques JA. Nitroreductases: Enzymes with Environmental Biotechnological and Clinical Importance. InCurrent Research, Technology and Education Topics in Applied Microbiology and Microbial Biotechnology; Mendez-Vilas, A., Ed.; Formatex: Badajoz, Spain, 2010:1008–1019.

Suter W, Hartmann A, Poetter F, Sagelsdorff P, Hoffmann P, Martus HJ. Genotoxicity assessment of the antiepileptic drug AMP397, an Ames-positive aromatic nitro compound. Mutat Res. 2002 Jul 25;518(2):181-94.

Umbuzeiro GA, Freeman H, Warren SH, Kummrow F, Claxton LD. Mutagenicity evaluation of the commercial product CI Disperse Blue 291 using dierent protocols of the Salmonella assay. Food and Chemical Toxicology 2005;43:49–56.

Tweats D, Bourdin Trunz B, Torreele E. Genotoxicity profile of fexinidazole--a drug candidate in clinical development for human African trypanomiasis (sleeping sickness). Mutagenesis. 2012 Sep;27(5):523-32.

Additional information

Bacteria reverse mutation

The mutagenic activity of Disperse Blue 366 (98.3%purity),was investigated in Salmonella typhimurium strains TA1535, TA1537, TA1538, TA98 and TA100. Strains were treated with Disperse Blue 366 by the Ames plate incorporation method at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system at 10% in standard co-factors. The dose range was determined in a preliminary toxicity assay and was 8 to 5000 μg/plate in the first experiment. The experiment was repeated on a separate day using different cultures of the bacterial strains and fresh chemical solutions. In this case, the dose range of Disperse Blue 366 was 312.5 to 5000 μg/plate. All solvent (DMSO) control plates gave counts of revertant colonies within the normal range. All positive control chemicals gave increases in revertants, both with and without the metabolising system, within expected ranges. Disperse Blue 366 did not cause a reduction in the growth of the bacterial lawn at any dose level in any of the strains of Salmonella. Disperse Blue 366 was, therefore, tested up to the maximum recommended dose of 5000 μg/plate.

A significant dose-related increase in the numbers of revertant colonies was recorded for all of the bacterial strains at doses ranging from 8 - 5000 μg/plate both with and without metabolic activation in both experiments. Disperse Blue 366 was found to exhibit evidence of marked mutagenic activity under the conditions of this test.

Disperse Blue 291 (commercial product: 30 to 50% dye content) was tested for mutagenic activity in the Salmonella assay with strains with different levels of nitroreductase andO-acetyltransferase (i.e., TA98DNP6, YG1024, and YG1041) as well as standard strains TA 1535, TA1537, TA1538, TA98 and TA100 and strains which provide more information on the base-pair substitution (TA 7001 to 7006). Disperse Blue 291 showed direct-acting mutagenic activity with all strains ofSalmonella typhimuriumtested, except forTA 1535.According to the classification of Claxton et al. (1991), the potency of this product can be considered moderate (10-100 revertants/µg). In the absence of S9, the nitroreduction is strongly related to the mutagenic activity, because the mutagenicity of Disperse Blue 291 was very low when tested with the strains lacking nitroreductase activity (TA98NR) and was increased with the nitroreductase overproducing strains, (YG1021 and YG1041). The same mutagenic pattern was observed for the acetyltransferase deficient and overproducing strains (TA98DNP6, YG1024, and YG1041) revealing also the importance of the acetyltransferase enzyme in the activation of Disperse Blue 291. Because of the remarkable increase in the response with the nitroreductase andO-acetyltransferase overproducing strain (YG1041), it is likely that the product of the nitroreductase is a substrate for theO-acetyltransferase.

In the presence of S9, the mutagenicity was slightly increased with TA98NR, TA98, YG1021, TA98DNP6, and YG1024 suggesting that, P450 enzymes also have a role in the activation of these compounds, besides the bacterial enzymes. This could be explained by the activation of other radicals of the molecule by the S9 enzymes, for example the —OCH3or the —N(CH2CH3)2of Disperse Blue 291, which however proved not to happen in studied with mammalian cells or in-vivo studies, as shown in the negative results with these studies.

Mammalian cell gene mutation

The study was performed to investigate the potential of the test item,Disperse Blue 165 (98.4%purity),to induce gene mutations at the HPRT locus in V 79 cells of the Chinese hamster in vitro. Two independent experiments were conducted both with and without an exogenous rat liver microsomal activation system (S9-mix). The compound was suspended in DMSO and tested at concentrations of 5, 10, 25, 50, 100, 250, 500, 1000 and 2000 μg/mL with and without metabolic activation. The concentration ranges were based on the results of preliminary tests for solubility and toxicity. The highest concentration showed no toxic effects with and without metabolic activation. Macroscopical visible precipitation of the test compound was observed down to a concentration of 50 μg/mL. In the presence of metabolic activation, a significant increase of the mutation frequency was observed only at a concentration of 50 μg/mL in the first main experiment. This effect was not dose-dependent, not reproduced in the second experiment and not three fold higher than the corresponding controls and therefore of no biological relevance. Up to the highest investigated dose no further increase in mutant colony numbers was obtained in two independent experiments. Appropriate reference mutagens used as positive controls showed a distinct increase in induced mutant colonies, thus indicating the sensitivity of the assay, and the efficacy of the S9-mix.

In conclusion, the substance does not induce gene mutations in the HPRT-test with V79 Chinese hamster cells, both in the presence as well as in the absence of a metabolic activation system, under the experimental conditions described. The test substance is therefore considered to be non-mutagenic in this HPRT assay.

Unscheduled DNA Synthesis

Disperse Blue 291 (96%purity)was tested for the ability to induce unscheduled DNA synthesis (UDS) in an in vivo rat hepatocyte assay. Male Fischer 344 rats were treated with a single oral dose of CI Disperse Blue 291 by gavage at 1250, or 2000 mg/kg body weight. The highest test dose, 2000 mg/kg was the limit test dose for a non-toxic test agent in this assay. Animals were killed and hepatocytes prepared four hours and twelve hours following administration of the chemical. Two independent experiments were carried out for each time point. Hepatocytes from treated rats were exposed to [³H]-thymidine and the amount of radioactivity incorporated into the nucleus [N] and an equal area of cytoplasm [C] determined by autoradiography. The cytoplasmic grain count was subtracted from that of the nucleus. The value obtained, the mean net nuclear grain count [N-C], is an index of UDS activity. In the respective testing laboratory, no negative control animal has shown a mean net nuclear grain count greater than zero. An [N-C] of more than zero in a treated animal is therefore considered indicative of a UDS response. Each experiment was validated by concurrent control treatments of rats with corn oil, the solvent for Disperse Blue 291 and with the carcinogens 2-acetylaminofluorene [2AAF] at twelve hours and N-nitrosodimethylamine [NDMA] or 6-p-dimethylaminophenylazobenzthiazole [6BT] at four hours. Solvent treated rats gave rise to mean net grain counts of less than zero, whilst hepatocytes from 2AAF, 6BT or NDMA treated animals had mean net nuclear grain counts of greater than +5. These data showed that background levels of UDS were normal and that the tester animals were responsive to known carcinogens requiring metabolic activation for genotoxic activity. Hepatocytes from Disperse Blue 291 treated animals were assessed for UDS at two dose levels of 1250 and 2000 mg/kg body weight. Treatments with Disperse Blue 291 in no case resulted in a mean net grain count greater than zero, at either time point. It is concluded that, when tested up to a limit dose of 2000 mg/kg body weight, the test sample of Disperse Blue 291 did not induce DNA repair (as measured by unscheduled DNA Synthesis) in hepatocytes from rats treated in vivo.

Mammalian erythrocyte micronucleus test

A study was performed to assess the potential of Disperse Blue 366 (98.3%purity) to produce damage to chromosomes or the mitotic apparatus of mice when administered by the oral route. The method used followed that described in the OECD Guidelines for Testing of Chemicals (1981) No. 474 “Genetic Toxicology: Micronucleus Test”. Following a preliminary range-finding study, groups of ten mice (five males and five females) were given a single oral dose of Disperse Blue 366 at the maximum practical dose (5000 mg/kg) in the main study. Animals were killed 24, 48 or 72 hours after test substance administration. Polychromatic erythrocytes were scored for the presence of micronuclei. Further groups of ten mice were treated with arachis oil B.P. or cyclophosphamide, to serve as vehicle and positive controls respectively.

There was no evidence of an increase in the incidence of micronucleated polychromatic erythrocytes in mice treated with Disperse Blue 366 when compared to the vehicle control groups. No statistical analysis was necessary. The positive control material produced a very marked increase in the number of micronucleated polychromatic erythrocytes. The test material, Disperse Blue 366, was considered to be non-clastogenic under the conditions of the test.

Justification for classification or non-classification

Based on the results of in vitro and in vivo testing, no classification for genotoxicity is required for the test substance according to CLP (EC 1272/2008) criteria.